Ion channels are membrane bound proteins that mediate fast neural dynamics by selectively controlling the flow of charged ions across membranes. Most channels are embedded within compositionally complex neuronal membranes, whose detailed composition play important roles in regulating channel functions. Membranes can regulate channels directly, through the binding of specific components to sites within channel structures, or indirectly, by impacting the biophysical and biochemical processes evolved to regulate channel functions in their native environment. A mechanistic understanding of how membrane composition impacts channel functions is vital because changes in neuronal membrane composition are associated with normal development and neurological disease. The goal of the proposed studies is to test three distinct mechanisms through which compositionally complex membranes regulate channel function. The working hypothesis, supported by past collaborative work of the Pl and Col, is that some channel functions are regulated by emergent properties of their embedding membranes that occur because these membranes are heterogeneous. Guided by extensive preliminary data, three specific aims will be pursued: 1) Measure the functional coupling of channel states to membrane domains, 2) Establish how membrane domains impact the binding of allosteric regulators, and 3) Identify the roles of membrane domains within the broader regulatory environment of neurons. The first aim experimentally tests a minimal model positing that single channel functions are allosterically regulated by domains within embedding membranes through tuning the availability of preferred local lipid environments. The second aim explores how the chemical potential of known allosteric regulators such as cholesterol and phosphoinositide lipids are impacted by the same thermodynamic parameters that control properties of membrane domains. The third aim investigates how membrane domains impact the sorting of enzymes that participate in protein palmitoylation and tyrosine phosphorylation regulatory pathways occurring at neuronal synapses. Experimental approaches draw on the PIs expertise using quantitative super-resolution fluorescence localization microscopy techniques and are combined with functional studies, theory, and simulation to test and refine mechanistic models of isolated and collective channel functions. The proposed work is innovative because it applies predictive models of membrane organization that are novel to both the channel and membrane domain communities. A broadly applicable framework for describing how domains modulate channel functions will drive advances in neuroscience by providing new insights into the functional basis for membrane changes with development and neurological disease, will motivate more effective and targeted treatments for neurological disease, and will connect the molecular-scale behaviors of channels to larger questions in neuroscience through the collective actions of lipids and membrane domains.